We thank S. Burkhart, C. Hofmann and Simone Gr&#228;&szlig;le for excellent technical assistance and are grateful to M. Ferg for help with data analysis. We also thank S. Rastegar for critical comments on the manuscript. We acknowledge funding by the Studienstiftung des deutschen Volkes (to MW), the DFG (DI913/4-1) and the Helmholtz Program BioInterfaces at KIT.

1. Prepare Working Dilution of Chemical Library
Predilute the compounds of the drug library to be tested (e.g. FDA approved drugs, ENZO Life Science) into E3 (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2) with a robotic liquid handling station (e.g. Multiprobe II, 8 needles with disposal tip adapter, PerkinElmer). Suitable concentrations are e.g. 40 &#956;g/ml in 3% DMSO, but this will depend on the type of library used. The Multiprobe II settings corresponding to the steps described below are indicated in Table 1. The protocol described in the table allows the preparation of four aliquot plates in parallel.
<ol>
	<li>Pipette 10 &#956;l aliquots of the stock library (2 mg/ml in DMSO) into deep well plates (e.g. 96-Well Storage Plate, Round Well, 0.8 ml, ABgene). Columns 1 and 12 should be left empty - these will receive the positive and negative within-plate controls.</li>
	<li>Dispense 490 &#956;l of E3 with 1% DMSO into the prepared aliquots of the library with the Multiprobe II. Dilution is performed with fixed needles without disposal tips.</li>
	<li>Add 10 &#956;l of DMSO into column 1 and 10 &#956;l of a 5 mM dexamethasone solution (in DMSO) to column 12 as within-plate controls. The concentration of DMSO in all wells is now 3%, the dexamethasone concentration in the positive control wells is 100 &#956;M in E3/3% DMSO.</li>
	<li>Seal the plate with DMSO resistant adhesive sealing sheets. Store the plates at -80 &#176;C until usage.</li>
</ol>
2. Breeding of Reporter Fish
Collect embryos from natural spawning of group matings between GRE:Luc transgenic fish. Raise embryos (not more than 60) in 9 cm Petri dishes containing E3 medium supplemented with 1 mg/ml of the fungicide methylene blue until 4 days post fertilization (dpf) in an incubator at 28 &#176;C. Change E3 medium regularly.
3. Preparation of Luciferase Medium (E3L)
Prepare an aqueous luciferin stock solution of 50 mM by adding dH2O to the vial containing the luciferin powder. This stock solution can be stored at -80 &#176;C for several months. Dilute the luciferin stock into E3 medium to a final concentration of 0.5 mM to obtain E3L medium.
4. Distribute Larvae into 96 Well Plates
<ol>
	<li>Prepare 1 ml pipette tips with wide bore by cutting off approximately 5 mm of the tip and briefly flaming the sharp edges.</li>
	<li>Pool larvae from several crosses by carefully pouring them from the Petri dishes into a beaker. Harvest about 50 larvae at a time by gently pouring them onto a sieve (pore size of 0.25 mm diameter) and immediately place the sieve into a small Petri dish (diameter 5.5 cm) filled with E3L medium.</li>
	<li>With a wide bore pipette tip, transfer 225 &#956;l of medium containing one larva each to the wells of a white 96 well plate (OptiPlate, PerkinElmer).</li>
	<li>Seal the plates with adhesive sealing sheets (e.g. TopSeal-A, PerkinElmer) and incubate them at 28 &#176;C overnight. This preincubation prevents recording of transient changes in bioluminescence immediately after addition of luciferin, a phenomenon that occurs also in cultured cells19.</li>
</ol>
5. Drug Treatment
<ol>
	<li>Remove the plate containing the working dilution of the library from the -80 &#176;C freezer about 4 hr before use. Gently vortex the plate and briefly spin it in a centrifuge to collect the liquid at the bottom of the plate.</li>
	<li>Remove adhesive sealing sheets from the larvae plates.</li>
	<li>With a hand-operated 96-channel pipetting device (e.g. Liquidator, Steinbrenner) pipette the drug solution up and down 3 times. Then aliquot 25 &#956;l of the working dilution of the chemical library into the wells containing the larvae (with the example above, this results in a final concentration of 4 &#956;g/ml in E3 with 0.3% DMSO). Prepare 10 (minimum: 5) replica plates with larvae per library plate.</li>
	<li>Seal the plates with the adhesive sealing sheets and label each plate with a barcode sticker.</li>
</ol>
6. Luminescence Recording
<ol>
	<li>Put the plates containing the larvae into the stacking units of an EnVision bioluminescence reader with enhanced luminescence sensitivity (PerkinElmer). Alternatively, use a robotized incubator system such as those employed for mammalian cell culture screening systems in combination with the reader.</li>
	<li>Record bioluminescence for two days using reader settings analogous to those described in Table 2 for the EnVision reader. Adapt the number of assay repeats to the number of plates in the run to match the required running time.</li>
	<li>After the end of the run, check the plates for the presence of dead larvae to evaluate general toxicity of the compounds.</li>
</ol>
7. Data Analysis
All data analysis is performed in the statistical programming environment R using custom scripts.
<ol>
	<li>AUC calculation 
	In order to identify compounds activating GC signaling regardless of their kinetic properties, determine the area under the curve (AUC) of the recorded luminescence traces (bioluminescence raw counts vs. time) as the screening parameter. AUCs are approximated with the trapezoidal rule (see Figures 2a and a&#39;).</li>
	<li>Normalization 
	Log transform the AUC values in order to ensure a higher normality of the data (Figures 2b and b&#39;). Then normalize the log transformed AUC values for each library plate with the robust Z-score method (see 20). This normalization results in values representing the number of standard deviations from the median of all data points. In this way, systematic errors, such as inter-run variability, are removed from the data. The data can now be visualized by plotting the mean robust Z-score of the replicas for each well (Figures 2c and c&#39;).</li>
	<li>Quality metrics
	<ol>
		<li>Heatmap 
		In order to visualize plate related effects, plot the data for each plate as a heatmap using e.g. the heatmap.2 function of the gplot package21. In this way, edge effects or compound carry-over can be easily detected (e.g. compare Figure 3a with 3b). Exclude sub-optimal plates from further analysis.</li>
		<li>ROC curve 
		To determine sensitivity and specificity of the assay, calculate a receiver operating characteristic (ROC) curve e.g. with the help of the ROC bioconductor package22 using the Z-score values determined in 7.2 (Figure 3c). Subsequently, determine the AUC of this curve. An AUC value close to 1 indicates high sensitivity and specificity of the assay.</li>
	</ol>
	</li>
	<li>Hit Identification 
	To optimize the cut-off value for the identification of active compounds, determine the Youden Index. To calculate the index, subtract the estimated true positive rate (TPR) from the false positive rate (FPR) of the ROC curve for increasing robust Z-score cut-off values. Take the robust Z-score with the highest Youden Index (TPR minus FPR) as the cut-off point. The TPR/FPR can be estimated by detection of positive control wells or of known active compounds within the library. When using positive control wells make sure that they match the anticipated hit strength.</li>
</ol>
8. Retesting
<ol>
	<li>Reevaluate positive hit compounds by treating larvae with serial dilutions of the compounds obtained from a different supplier, using the same recording set-up described above. True hits should induce luminescence also in this assay, ideally in a dose dependent manner.</li>
</ol>

<ol>
	<li>Norris, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vertebrate Endocrinology. , (2007).</li><li>Kassel, O., Herrlich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+the+glucocorticoid+receptor+and+other+transcription+factors:+molecular+aspects.">Crosstalk between the glucocorticoid receptor and other transcription factors: molecular aspects.</a> Mol Cell Endocrinol. 275, 13-29 (2007).</li><li>Baschant, U., Tuckermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+glucocorticoid+receptor+in+inflammation+and+immunity.">The role of the glucocorticoid receptor in inflammation and immunity.</a> J Steroid Biochem Mol Biol. 120, 69-75 (2010).</li><li>Schacke, H., Docke, W. D., Asadullah, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+involved+in+the+side+effects+of+glucocorticoids.">Mechanisms involved in the side effects of glucocorticoids.</a> Pharmacol Ther. 96, 23-43 (2002).</li><li>De Bosscher, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Glucocorticoid+Receptor+modulators.">Selective Glucocorticoid Receptor modulators.</a> J Steroid Biochem Mol Biol. 120, 96-104 (2010).</li><li>Kaufman, C. K., White, R. M., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetic+screening+in+the+zebrafish+embryo.">Chemical genetic screening in the zebrafish embryo.</a> Nat Protoc. 4, 1422-1432 (2009).</li><li>Peal, D. S., Peterson, R. T., Milan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+screening+in+zebrafish.">Small molecule screening in zebrafish.</a> J Cardiovasc Transl Res. 3, 454-460 (2010).</li><li>Casals-Casas, C., Desvergne, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+disruptors:+from+endocrine+to+metabolic+disruption.">Endocrine disruptors: from endocrine to metabolic disruption.</a> Annu Rev Physiol. 73, 135-162 (2011).</li><li>Grun, F., Blumberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+the+case+for+obesogens.">Minireview: the case for obesogens.</a> Mol Endocrinol. 23, 1127-1134 (2009).</li><li>Odermatt, A., Gumy, C., Atanasov, A. G., Dzyakanchuk, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+glucocorticoid+action+by+environmental+chemicals:+potential+mechanisms+and+relevance.">Disruption of glucocorticoid action by environmental chemicals: potential mechanisms and relevance.</a> J Steroid Biochem Mol Biol. 102, 222-231 (2006).</li><li>Odermatt, A., Gumy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoid+and+mineralocorticoid+action:+why+should+we+consider+influences+by+environmental+chemicals.">Glucocorticoid and mineralocorticoid action: why should we consider influences by environmental chemicals.</a> Biochem Pharmacol. 76, 1184-1193 (2008).</li><li>Lohr, H., Hammerschmidt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+in+endocrine+systems:+recent+advances+and+implications+for+human+disease.">Zebrafish in endocrine systems: recent advances and implications for human disease.</a> Annu Rev Physiol. 73, 183-211 (2011).</li><li>Schaaf, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+functional+glucocorticoid+receptor+beta-isoform+in+zebrafish.">Discovery of a functional glucocorticoid receptor beta-isoform in zebrafish.</a> Endocrinology. 149, 1591-1599 (2008).</li><li>Schaaf, M. J., Chatzopoulou, A., Spaink, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+system+for+glucocorticoid+receptor+research.">The zebrafish as a model system for glucocorticoid receptor research.</a> Comp Biochem Physiol A Mol Integr Physiol. 153, 75-82 (2009).</li><li>Alsop, D., Vijayan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+stress+axis:+molecular+fallout+from+the+teleost-specific+genome+duplication+event.">The zebrafish stress axis: molecular fallout from the teleost-specific genome duplication event.</a> Gen Comp Endocrinol. 161, 62-66 (2009).</li><li>Alsop, D., Vijayan, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+programming+of+the+corticosteroid+stress+axis+during+zebrafish+development.">Molecular programming of the corticosteroid stress axis during zebrafish development.</a> Comp Biochem Physiol A Mol Integr Physiol. 153, 49-54 (2009).</li><li>Schoonheim, P. J., Chatzopoulou, A., Schaaf, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+an+in+vivo+model+system+for+glucocorticoid+resistance.">The zebrafish as an in vivo model system for glucocorticoid resistance.</a> Steroids. 75, 918-925 (2010).</li><li>Weger, B. D., Weger, M., Nusser, M., Brenner-Weiss, G., Dickmeis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Chemical+Screening+System+for+Glucocorticoid+Stress+Hormone+Signaling+in+an+Intact+Vertebrate.">A Chemical Screening System for Glucocorticoid Stress Hormone Signaling in an Intact Vertebrate.</a> ACS Chem Biol. 7, (2012).</li><li>Hirota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+biology+approach+reveals+period+shortening+of+the+mammalian+circadian+clock+by+specific+inhibition+of+GSK-3beta.">A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta.</a> Proc Natl Acad Sci U S A. 105, 20746-20751 (2008).</li><li>Birmingham, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+methods+for+analysis+of+high-throughput+RNA+interference+screens.">Statistical methods for analysis of high-throughput RNA interference screens.</a> Nat Methods. 6, 569-575 (2009).</li><li>Warnes, G. R., Bolker, B., Bonebakker, L., Gentleman, R., Liaw, W. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> gplots: Various R programming tools for plotting data. 2.11.0, (2012).</li><li>Carey, V., Redestig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ROC: utilities for ROC, with uarray focus. , (2013).</li><li>Fan, F., Wood, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+assays+for+high-throughput+screening.">Bioluminescent assays for high-throughput screening.</a> Assay Drug Dev Technol. 5, 127-136 (2007).</li><li>Graf, S. F., Hotzel, S., Liebel, U., Stemmer, A., Knapp, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+fluidic+sorting+system+for+automated+Zebrafish+egg+sorting+into+multiwell+plates.">Image-based fluidic sorting system for automated Zebrafish egg sorting into multiwell plates.</a> J Lab Autom. 16, 105-111 (2011).</li><li>Letamendia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+automated+high-throughput+system+for+zebrafish+in+vivo+screenings.">Development and validation of an automated high-throughput system for zebrafish in vivo screenings.</a> PLoS ONE. 7, e36690 (2012).</li></ol>

We have nothing to disclose.

We present here the workflow and data analysis for a chemical screen measuring GC activity in vivo using the GRIZLY assay18. The assay has quality control characteristics and performance measures that are comparable with conventional cell based screens, an important advantage for its use in high-throughput settings. In addition, however, the in vivo assay detects also compounds that are not accessible to in vitro screens. This is exemplified by the presence of the prohormone pregnenolone among the hits. Thus, the GRIZLY assay extends the scope of screens aimed at GC signaling activity.
The importance of the detection of in vivo effects with our assay is also illustrated by results we obtained with the organotin pollutants DBT and TBT18. DBT, but not TBT, has been shown to inhibit GC signaling in mammalian cell culture, and we observed the same in zebrafish cell cultures expressing the GRE:Luc reporter. Importantly however, in the GRIZLY assay, TBT showed inhibitory activity on the GC pathway, as it can be converted to DBT by the larval metabolism. This inhibition was observed already at environmentally relevant concentrations of TBT. These results further illustrate the potential of the GRIZLY assay in detecting compound effects based on inter-organ cross-talk and metabolic modifications of the compounds in the living animal. They also highlight the application potential of the GRIZLY assay for environmental monitoring. Thus, endocrine disruptor effects can be studied at the level of receptor signaling, where the disruptor interferes with GC signaling activity induced by a GR agonist such as dexamethasone. Another possibility is to study compound effects on pregnenolone-stimulated GC synthesis. The high throughput quality of the assay should permit the rapid screening of environmental sample libraries.
The use of luciferase as a reporter gene allows for a high dynamic range of detection of signaling activity23. Indeed, the sensitivity of the assay makes it possible to detect osmotic stress-induced GC production from single larvae18. The high temporal resolution achieved with the luciferase reporter allows us to follow signaling activities over time, which makes it possible to analyze also the kinetics of the data.
A potential drawback for some applications is the limited suitability for spatial monitoring of this reporter system. Fluorescent reporter systems might be better suited for assays that require monitoring of certain areas of the larvae. However, such assays might suffer from lower sensitivities due to background effects caused by the excitation light, which also poses limits on the detection of fluorescent compounds. Additionally, they might provide less kinetic resolution because of the generally higher stability of fluorescent proteins23. Furthermore, they present challenges in terms of imaging equipment, data analysis and data storage that might limit their use for smaller research labs.
The set-up of the GRIZLY assay as a microtiter plate based assay allows for easy integration into typical screening workflows. The assay is easily applicable also in smaller research labs due to its simple handling and data analysis. At the same time, it allows for a substantial degree of automation, e.g. automated drug application by pipetting robots or automated distribution of embryos by embryo sorting devices24,25. The simple readout does not require automated screening microscopes or sophisticated image analysis software, yet provides a rich set of data on temporal and quantitative aspects of the studied signaling pathway.
In summary, we present a step-by-step protocol for a relatively inexpensive, robust and easy-to-handle chemical screening assay for GC signaling activity in vivo and in real time. The assay allows the determination of in vivo effects of compounds on GC signaling not detectable in cell culture based assays. Among the many applications for the assay are e.g. the determination of genetic effects on glucocorticoid signaling, environmental monitoring of endocrine disruptor effects on glucocorticoid synthesis and signaling activity, and the screening of compounds for unwanted effects on GC signaling or for novel in vivo modulators of this important signaling pathway.

Glucocorticoids (GCs) are steroid hormones produced by the adrenal gland which play important roles during the stress response and in the regulation of metabolism1. GCs bind cytoplasmic glucocorticoid receptors (GRs) of the nuclear receptor superfamily which, upon binding, translocate into the nucleus2. Here, they can for example repress transcription by interfering with other transcription factors (transrepression) or activate gene transcription via glucocorticoid response elements (GREs, transactivation). Due to their anti-inflammatory properties, both natural and artificial GCs are widely used drugs in the treatment of a number of diseases, such as asthma or arthritis3. However, especially long-term use of GCs can lead to severe side effects, including diabetes and glaucoma4. Therefore, novel compounds targeting GC signaling with potentially more beneficial treatment efficiency and tolerability are highly sought after. Importantly, ligand effects observed in cultured cells can differ from those seen in vivo5. Such effects might bias results obtained with conventional cell culture based pharmaceutical screening assays. Chemical in vivo screening as enabled by the zebrafish model has recently come into focus, as it allows the determination of effects not detectable in cell culture6,7.
Hormonal signaling pathways can also be affected by environmental pollutants. So-called endocrine disrupting chemicals (EDCs) affect various hormone regulated processes8. Thus, reproduction and sexual differentiation of aquatic organisms can be modulated by substances with estrogen-like activities. Recently, concerns have been raised that metabolic disorders might be linked to EDCs in the environment9. One pathway targeted by such &#34;metabolic disruption&#34; is the glucocorticoid pathway, which has also been implicated in xenobiotics effects on development and immune function10,11. However, compared with the large amount of information available on compounds interfering with sex steroid hormone action, relatively little is known about endocrine disruption effects mediated via the GR. Therefore, tools are needed that allow monitoring pollutant effects on GC signaling in vivo.
The zebrafish has long been a popular model in developmental biology and has more recently also attracted researchers from other fields, including endocrinology12. Compared with other teleosts, the GC signaling system of zebrafish is more similar to that of mammals, since the zebrafish genome contains only one GR gene as opposed to the duplicated receptors in many other fish species13-15. In addition, the hypothalamic-pituitary-adrenal axis is already functional in 5 day old zebrafish larvae, which increase endogenous GC production in response to stressors14,16-18.
We have recently generated a transgenic zebrafish line, GRE:Luc, which allows for the monitoring of GC signaling activity in vivo and in real-time18. The line carries a luciferase reporter gene construct under control of a minimal TATA box promoter and four concatemerized GREs (Figure 1a). GC induced bioluminescence can be measured from single GRE:Luc larvae in 96 well microtiter plates in vivo over prolonged periods of time. This GRIZLY assay (for &#34;Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY&#34;) can be used in a number of different research fields, such as stress research, environmental monitoring, and pharmacological screens18. We were able to detect the endogenous rise in cortisol after osmotic stress from single larvae and could follow the maturation of the response during development. Furthermore, we could monitor the effects on GC signaling of organotins that require metabolization by the larva. Importantly, the line was able to detect these effects at environmentally relevant concentrations. Finally, in a pilot screen the assay sensitively and specifically detected compounds with GC activity from a chemical library, including one compound that stimulated endogenous cortisol production in the larvae. Here, we describe a detailed protocol for chemical screens using the GRIZLY assay.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Buffer composition + reagents&lt;/strong&gt;</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Carl Roth GmbH Co KG</td><td>A994.2</td><td></td></tr><tr><td>FDA approved drug library</td><td>Enzo Life Sciences</td><td>BML-2841-0100</td><td></td></tr><tr><td>Luciferin</td><td>Biosynth</td><td>L-8220</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>Methylene Blue</td><td>Sigma-Aldrich</td><td>M9140</td><td></td></tr><tr><td>E3</td><td>N/A</td><td>N/A</td><td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Instruments&lt;/strong&gt;</td></tr><tr><td>Multiprobe II</td><td>PerkinElmer</td><td></td><td>8 channel, equipped with disposable tip adaptor</td></tr><tr><td>Liquidator 96</td><td>Steinbrenner Laborsysteme</td><td></td><td>hand-operated 96-channel pipette</td></tr><tr><td>EnVision XCite Multilabel Plate Reader</td><td>PerkinElmer</td><td></td><td>Equipped with stacker automation, temperature control, barcode reader and enhanced luminescence detector</td></tr><tr><td colspan="4">&lt;strong&gt;Plasticware + consumables&lt;/strong&gt;</td></tr><tr><td>96-Well Storage Plate</td><td>ABgene</td><td>AB-0765</td><td>round well, 0.8 ml</td></tr><tr><td>Cover films,</td><td>ratiolab</td><td>6018412</td><td>self adhesive, DMSO resistent</td></tr><tr><td>Pipette tips</td><td>Steinbrenner Laborsysteme</td><td>LRF-200L</td><td>for liquidator 96, 200 &amp;mu;l, low retention</td></tr><tr><td>TopSeal-A</td><td>PerkinElmer</td><td>6005185</td><td></td></tr><tr><td>OptiPlate-96</td><td>PerkinElmer</td><td>6005299</td><td>white opaque 96-well microplate</td></tr><tr><td>Barcode Labels</td><td>PerkinElmer</td><td>1608182</td><td></td></tr><tr><td>filtered polypropylene IsoTip pipette tips</td><td>Corning</td><td>S058.4809</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Animals&lt;/strong&gt;</td></tr><tr><td>GRE:Luc fish</td><td>N/A</td><td>ZDB-TGCONSTRCT-120920-1</td><td>available at the European Zebrafish Resource Centre EZRC, Karlsruhe</td></tr></tbody></table>

a chemical screening procedure for glucocorticoid signaling with a zebrafish larva luciferase reporter system

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

In a previous publication18, we screened a library of 640 FDA approved drugs in a pilot screen to evaluate the performance of the GRIZLY assay. This library contained 12 bona fide GCs, thereby allowing us to determine quality measures for sensitivity and specificity of the assay.
Figure 2 shows typical examples of the raw data analysis and normalization taken from this screen. A typical result from a dexamethasone control well is depicted in Figure 2a, illustrating the temporal information given by the data. A peak in bioluminescence that occurs at about 12 hr after the treatment slowly decreases over the following 36 hr of measurement. Figure 2a&#39; highlights the approximation of the AUC value using the trapezoidal method. This calculation is carried out for all wells and each technical repeat. The result of the log transformation is shown in Figures 2b and b&#39;. Here the mean of the positive controls are plotted in a normal Q-Q plot. Log transformation leads to a greater approximation of the data points to the theoretical normally distributed values indicated by the red diagonals. A transformation to achieve normality of the data increases the statistical power of the subsequent analyses. Finally, Figures 2c and 2c&#39; show the effect of robust Z-score normalization on plate-to-plate variability: The different baseline values obtained with different plates (Figure 2c) are normalized in the robust Z-score plot in Figure 2c&#39;.
The robust Z-score values can be used to identify systematic errors in the data by visualizing the distribution of the hits across the plates. Figure 3a shows one example of a heatmap of a library plate, in which the robust Z-score values are plotted color-coded into the well positions. One easily identifies column 12 with the in-plate positive controls. Positive scoring library compounds are seen in well F8 and, albeit weaker, E2. Figure 3b shows a plate in which a systematic error is present. One observes a series of compounds with decreasing Z-score values in rows A and E over several columns. None of the compounds in these wells was tested positive in the re-test. Since the wells with this decreasing GRIZLY test activity are placed along the path the pipetting robot takes when aliquoting the library plates, this pattern is indicative of carry-over of a positive compound during the automated pipetting process.
The robust Z-score normalization together with the presence of known GCs in the library also enabled us to calculate a receiver operating characteristic (ROC) curve for the screen18. Figure 3c shows a plot of the estimated true positive rate against the estimated false positive rate for different robust Z-score cut-off values. The estimated true positive rate is defined as the percentage of true positive hits (here, the known glucocorticoids present in the library) that are found at a given robust Z-score cut-off value. The corresponding estimated false positive rate indicates the percentage of non-positive compounds hit at this cut-off value. The AUC of this curve is 0.95, indicating a high sensitivity and specificity of the screen and excellent assay performance. The AUC curve was also used to estimate the optimal cut-off value for hit identification by calculating the Youden index for different robust Z-scores. We identified the robust Z-score of 1.49 as having the highest Youden index. This cut-off value is indicated in Figure 2c&#39; as a black line separating the hits from the bulk of the compounds.
In our screen18, we were able to identify nearly all known GCs present in the library. Only three of the 12 bona fide GCs were not detected. In the case of melengestrol acetate, this was a false negative finding, since this compound tested positive in the re-test at a higher concentration than the one used in the library. The other two GCs were negative also in the re-test. Corticosterone is the major GC in rodents, but not in fish or humans1, while the prodrug prednisone might not be metabolized well by the larval system. Interestingly, also two drugs not annotated as GCs were identified in the screen, of which one could not be confirmed in the re-test (spironolactone, a mineralocorticoid receptor antagonist). The other compound, pregnenolone, is a precursor in steroid biosynthesis, which is converted to cortisol by the adrenal gland. Indeed, treatment with pregnenolone stimulated cortisol production in the larvae18. All these results confirm the high performance of the assay: only one false negative and one false positive compound were among the primary screen results, and 10 of the 11 confirmed compounds with GC signaling stimulating activity were already identified in the primary screen.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50439/50439fig1.jpg" /> 
Figure 1. General principle of the assay and work flow of the screening procedure. a) Scheme of the GRIZLY assay reporter construct. Luciferase (yellow) expression is controlled by a minimal promoter (Pmin, orange) and four concatemerized GRE elements ((GRE)4, white), which are bound by ligand-activated GR homodimers (GR, blue). Red boxes indicate Tol2 transposase sites that facilitate integration of the construct into the genome. The pink box represents a poly-adenylation site (pA). Transgenic larvae carrying this construct are placed in opaque 96 well microtiter plates for bioluminescence measurements. b) Scheme of the luciferase reaction. Luciferase catalyzes the oxidation of luciferin to oxyluciferin, which leads to light emission (h&#957;). The reaction requires also ATP and Mg2+, which is provided by the larva. PPi, pyrophosphate. c) Workflow of the screen. Working stock dilutions are prepared from an FDA approved drug library in 96 well plates, one column containing a negative within-plate control (DMSO only, green) and one column containing a positive control (Dexamethasone, red) (library design). GRE:Luc larvae are distributed in 96 well plates (5-11 replicates) and treated with the library compounds (drug application). Bioluminescence traces are recorded with a bioluminescence reader for two days (data collection). Bioluminescence traces are integrated (AUC calculations), log transformed and robust Z-score normalized (normalization). Normalized data are used for determining quality metrics and for hit identification (data analysis). Chosen hits are retested for dose-dependent activity in the assay (retest). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50439/50439fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/50439/50439fig2.jpg" /> 
Figure 2. Data analysis illustrated with data from a screen of a FDA approved drug library. a) Representative trace from a positive control well. a&#39;) The area under the curve (AUC) is approximated with the trapezoid rule. The trapezoids used for the calculation are shown in different shades of red. b) Normal Q-Q plot of raw AUC values of the positive control wells (y-axis) vs. a standard normal population (x-axis) before log transformation. b&#39;) Normal Q-Q plot of the AUC values after log transformation. A normal distribution is indicated by the linearity of the log transformed data points. c) Plot of log transformed raw data for all compounds tested in the screen. Blue, bona fide glucocorticoids; grey, all other compounds. c&#39;) Plot of screen data after robust Z-score normalization. Systematic errors such as inter-plate variations have been removed from the data. Red lines indicate the mean &#177; s.e.m. of positive within-plate controls. Black line: hit identification cut-off value as determined by Youden-Index optimization (Figure 3). Reproduced with permission from14, 2012 ACS. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50439/50439fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="4.5in" src="/files/ftp_upload/50439/50439fig3.jpg" /> 
Figure 3. Screen results. a) Heatmap of screen results. Robust Z-score values of library compounds are plotted into the respective well positions. The positive controls in column 12 as well as two positive hit compounds in wells F8 and E2 are visible. b) Heatmap of a plate showing carry-over of a positive compound during library preparation with a pipetting robot. A gradient of activity is visible along the pipetting path taken by the robot. c) Receiver operating characteristic (ROC) curve for the screen. The estimated true positive rates (left-hand y-axis) and false positive rates (x-axis) are plotted for increasing robust Z-score cut-off values (color coded, right-hand y-axis). The AUC value of the resulting curve is close to 1, indicating good assay performance. Reproduced with permission from 14, 2012 ACS. d) Table showing compounds active (yellow) or inactive (blue) in the primary screen (prim.) or in the retest. Bona fide glucocorticoids were not always retested (white). Redrawn with permission from14, 2012 ACS. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/50439/50439fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">General settings: </td>
		</tr>
		<tr>
			<td colspan="4">Step 1.1</td>
		</tr>
		<tr>
			<td>Procedure </td>
			<td>type:</td>
			<td colspan="2">single liquid</td>
		</tr>
		<tr>
			<td> </td>
			<td>mode:</td>
			<td colspan="2">blow out</td>
		</tr>
		<tr>
			<td> </td>
			<td>dispenses per aspirate:</td>
			<td colspan="2">1</td>
		</tr>
		<tr>
			<td> </td>
			<td>optimize for speed:</td>
			<td colspan="2">Yes</td>
		</tr>
		<tr>
			<td> </td>
			<td>air gaps system:</td>
			<td colspan="2">10 &#956;l</td>
		</tr>
		<tr>
			<td> </td>
			<td>transport:</td>
			<td colspan="2">0</td>
		</tr>
		<tr>
			<td>Flush/Wash </td>
			<td colspan="3" width="426">No </td>
		</tr>
		<tr>
			<td>Aspirate </td>
			<td>Type of setting </td>
			<td>Value </td>
			<td>remarks </td>
		</tr>
		<tr>
			<td> </td>
			<td>Position</td>
			<td>B7, B10, B13, B16</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Aspirate Vol.</td>
			<td>10 &#956;l</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Speed</td>
			<td>200 &#956;l/sec</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Liquid tracking</td>
			<td>60%</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Aspirate Height</td>
			<td>Labware default</td>
			<td>22.28</td>
		</tr>
		<tr>
			<td>Dispense </td>
			<td>Type of setting </td>
			<td>Value </td>
			<td>remarks </td>
		</tr>
		<tr>
			<td> </td>
			<td>Positions</td>
			<td>D7, D10, D13, D16</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Dispense Vol.</td>
			<td>10 &#956;l</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Speed</td>
			<td>200 &#956;l/sec</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Liquid tracking</td>
			<td>100 &#956;l</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Dispense Height</td>
			<td>Labware default</td>
			<td>17.01</td>
		</tr>
		<tr>
			<td colspan="4">Step 1.2 </td>
		</tr>
		<tr>
			<td>Procedure </td>
			<td>type: </td>
			<td colspan="2">reagent </td>
		</tr>
		<tr>
			<td> </td>
			<td>mode:</td>
			<td colspan="2">waste</td>
		</tr>
		<tr>
			<td> </td>
			<td>dispenses per aspirate:</td>
			<td colspan="2">3</td>
		</tr>
		<tr>
			<td> </td>
			<td>optimize for speed:</td>
			<td colspan="2">Yes</td>
		</tr>
		<tr>
			<td> </td>
			<td>air gaps system:</td>
			<td colspan="2">15 &#956;l</td>
		</tr>
		<tr>
			<td> </td>
			<td>transport:</td>
			<td colspan="2">0</td>
		</tr>
		<tr>
			<td colspan="2">Flush/Wash </td>
			<td colspan="2">No </td>
		</tr>
		<tr>
			<td>Aspirate </td>
			<td>Type of setting </td>
			<td>Value </td>
			<td>remarks </td>
		</tr>
		<tr>
			<td> </td>
			<td>Position</td>
			<td>A4</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Aspirate Vol.</td>
			<td>3 x 490 &#956;l</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Air gaps</td>
			<td>15 and 0</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Speed</td>
			<td>200 &#956;l/sec</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Liquid tracking</td>
			<td>400%</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Aspirate Height</td>
			<td>Liquid surface</td>
			<td> </td>
		</tr>
		<tr>
			<td>Dispense </td>
			<td>Type of setting </td>
			<td>Value </td>
			<td>remarks </td>
		</tr>
		<tr>
			<td> </td>
			<td>Position</td>
			<td>D7, D10, D13, D16</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Dispense Vol.</td>
			<td>490 &#956;l</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Air gaps</td>
			<td>15 and 0</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Speed</td>
			<td>200 &#956;l/sec</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Liquid tracking</td>
			<td>100%</td>
			<td> </td>
		</tr>
		<tr>
			<td> </td>
			<td>Dispense Height</td>
			<td>Labware default</td>
			<td>17.01</td>
		</tr>
	</tbody>
</table>
Table 1. Settings for Multiprobe II. The protocol in Table 1 describes the preparation of 4 plates (each 96 wells). The plates are located on plate adapters in the working area at positions specified by the robot (e.g. B7, B10, B13, B16).
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">General settings:</td>
		</tr>
		<tr>
			<td colspan="2">Number of assay repeats </td>
			<td colspan="2">dependent on the length of the run </td>
		</tr>
		<tr>
			<td colspan="2">Number of plates</td>
			<td colspan="2">unlimited</td>
		</tr>
		<tr>
			<td colspan="2">Temperature control</td>
			<td colspan="2">28 &#176;C</td>
		</tr>
		<tr>
			<td colspan="2">Protocol </td>
			<td colspan="2">Calculations </td>
		</tr>
		<tr>
			<td> </td>
			<td colspan="3">US Lum 96 (cps) read</td>
		</tr>
		<tr>
			<td> </td>
			<td>Measurement time</td>
			<td>2.5 sec</td>
			<td rowspan="3">Crosstalk correction</td>
		</tr>
		<tr>
			<td> </td>
			<td>Distance between plate and detector</td>
			<td>0 mm</td>
		</tr>
		<tr>
			<td> </td>
			<td>Glow (CT2) correction factor</td>
			<td>0 %</td>
		</tr>
	</tbody>
</table>
Table 2. EnVision settings used for the screen.

Watch this Scientific Journal Video about A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System at JoVE.com

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System | Biology  | Video

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

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11.9K Views.  Karlsruhe Institute of Technology - Campus North. We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Video: A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Developmental Biology

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Immunology and Infection

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Cancer Research

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Neuroscience

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Summary

Explore More Videos

Chapters in this video

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Development of automated imaging and analysis for zebrafish chemical screens.

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites